In any type of assay it is standard accepted practice to include controls which the operator can use to confirm the correct functioning of not only the assay system as a whole but also the correct/proper addition and/or functioning of various reagents, samples and/or equipment. Depending on their function, controls for use in assays, including immunological assays, may be referred to as positive or negative controls.
A number of assays require the addition of red blood cells; these assays include, for example, assays for the blood typing of red blood cell samples as performed routinely in blood donation testing centers and transfusion laboratories worldwide. Tests of this type include ABO and RhD grouping; comprehensive blood type testing may include (but is not limited to) the detection of Rh, Kell, Duffy, MNS and/or Kidd antigens.
Blood typing testing may be carried out as an agglutination test in a test tube. More recently tests of this type have been carried out using solid-phase microplate and column agglutination technologies (aka Gel, CAT). Current state-of-the-art includes commercially available systems such as Beckman Coulter PK7300, Immucor Capture-R, and BioRad ID-System and Ortho Clinical Diagnostics BioVue and ID-MTS systems, and although many other variations are now available, they are very similar in principle to the systems mentioned above.
Assays such as those described above rely on the successful addition of red blood cells. In most cases, the volume and quantity of the consumables/samples is large enough that the successful addition of samples and reagents (such as blood/red blood cells) can be recorded by simple observation. In the case of the addition of red blood cells to assays, their shape, form, density and colour makes them easily detectable. Some systems may use haemagglutination as the end point read out (interpreted by for example photographic technology and software) and the addition of red blood cells is easy to determine not least because the volumes and quantities of consumables (samples and the like) is large enough to permit easy visual detection of red blood cells but also because the haemagglutination process in itself provides evidence of red blood cell addition.
However, miniaturisation of assays presents a need for methods of detecting red blood cells which do not rely (solely) on visual identification. However, any method must respect the diversity of antigens expressed by red blood cells. In humans, red blood cells from persons with different genetic backgrounds and/or from different geographic locales, may express different antigen profiles. As such, any method which is to be exploited as a means to detect red blood cells and/or serve as a control test to confirm the addition of red blood cells, must reliably detect red blood cells of any type.
The presence of antigens (including blood group antigens) on the surface of red blood cells forms the basis of many immunological tests including, for example blood typing assays which may use non-agglutination protein microarrays, in which an immobilised antibody binds to an antigen on the surface of the red blood cells, and the presence of red blood cells so immobilised is detected (J S Robb et al 2006). Antibody microarray technology can also be used to phenotype red blood cells by detecting complex mixtures of antigens on cell surfaces (C. J. Campbell et al 2006). The antigens expressed by red blood cells are both sugar antigens, which tend to be well presented and easily accessible, and protein peptide antigens, which are epitopes of transmembrane or membrane linked proteins and therefore buried and held more closely to the cell surface, and these were successfully differentiated using the correct choice of antibodies.
With the advent of new miniaturised technologies, such as microarrays, surface plasmon resonance, and any other forms of assay/method and/or system which require the use or addition of red blood cells, it is desirable to provide a test or means which can reliably, repeatedly and consistently detect red blood cells of all types. In particular, it is desirable to provide red blood cell detection methods suitable for use in methods and assays/systems which do not (or cannot) use visual detection methods to detect red blood cells. Moreover, it is advantageous to provide a control test which can reliably, consistently and repeatedly report the addition of red blood cells to an assay or assay system.